Abstract

During development of the vertebrate hindbrain, the neuroepithelium becomes subdivided into seven morphological units, known as rhombomeres. It is necessary that rhombomeres have sharp, well-defined boundaries, which are established from initially rough gene expression domains during early hindbrain segmentation. The mechanisms involved in early hindbrain segmentation that create sharp segment borders are not well understood. There is evidence to suggest that both regulation of cell identity and Eph/ephrin-mediated cell sorting are required for establishing sharp interfaces between rhombomeres. This thesis investigates the extent to which identity regulation contributes to hindbrain border sharpening in zebrafish. I created a new zebrafish reporter line by CRISPR/Cas9-mediated reporter integration at the egr2b locus, which enables cell identity and cell intermingling to be visualised in live embryos during border sharpening. This new reporter line indicates a contribution of cell identity regulation to border sharpening. I also demonstrate that the contribution of cell identity switching to border refinement is greater in cases where cell intermingling is increased by perturbed Eph/ephrin signalling. To help study the role of Eph/ephrin signalling in border sharpening, I have also created a novel EphrinB3b mutant. The thesis also investigates the mechanisms of identity regulation by community effects and discusses their contribution to border refinement by identity respecification; community effects are suspected to help overcome noise in early gene induction through spatial averaging and thus help establish regions of homogeneous gene expression. The ability of candidate genes to non cell-autonomously regulate the identity of neighbouring cells in the hindbrain is investigated. Of particular focus is the potential involvement of retinoic acid (a morphogen involved in specification of anteroposterior identity) and segmentally-expressed Cyp26 enzymes involved in its metabolism. Analysis of mosaic embryos is used to compare the ability of isolated cells and clustered groups of cells to maintain a different identity to their surroundings. Results presented here are consistent with segmental regulation of retinoic acid signalling contributing to border sharpening.

Type:

Thesis
(Doctoral)

Title:

Investigating the Roles of Cell Identity Regulation and Eph/ephrin Signalling in Early Hindbrain Segmentation